AtMYB50 regulates root cell elongation by upregulating PECTIN METHYLESTERASE INHIBITOR 8 in Arabidopsis thaliana

Plant root development involves multiple signal transduction pathways. Notably, phytohormones like auxin and cytokinin are well characterized for their molecular mechanisms of action. Reactive oxygen species (ROS) serve as crucial signaling molecules in controlling root development. The transcription factor, UPBEAT1 (UPB1) is responsible for maintaining ROS homeostasis at the root tip, influencing the transition from cell proliferation to differentiation. While UPB1 directly regulates peroxidase expression to control ROS homeostasis, it targets genes other than peroxidases, suggesting its involvement in root growth through non-ROS signals. Our investigation focused on the transcription factor MYB50, a direct target of UPB1, in Arabidopsis thaliana. By analyzing multiple fluorescent proteins and conducting RNA-seq and ChIP-seq, we unraveled a step in the MYB50 regulatory gene network. This analysis, in conjunction with the UPB1 regulatory network, demonstrated that MYB50 directly regulates the expression of PECTIN METHYLESTERASE INHIBITOR 8 (PMEI8). Overexpressing PMEI8, similar to the MYB50, resulted in reduced mature cell length. These findings establish MYB50 as a regulator of root growth within the UPB1 gene regulatory network. Our study presents a model involving transcriptional regulation by MYB50 in the UPB1 regulated root growth system and sheds light on cell elongation via pectin modification.


Introduction
Plant roots are important organs that support the entire plant body, absorb water and nutrients from the soil, and respond to environmental changes.To achieve optimal root architecture, the root developmental mechanism is strictly controlled.Maintaining the balance of transition from cell proliferation to differentiation at the root tip is a key to coordinating primary root growth.Cell proliferation occurs in the meristematic zone that includes the stem cell niche.Once the cell leaves the meristematic zone, it stops proliferating but starts rapidly elongating in the elongation zone.After the cells fully elongate, the cells mature into their final states and function in the maturation zone.Maintaining the precise balance of cell status among these zones is vital for root growth.For this process, the transcriptional network is important [1,2] and almost all hormones are involved in the transcriptional network for regulating the balance between cell proliferation and differentiation at the root tips [3,4].
In addition to hormones, reactive oxygen species (ROS) also act as regulators to maintain the balance of cell function in the root tip.ROS, such as superoxide, hydrogen peroxide, and hydroxyl radicals, are thought of as molecules that are harmful to the cells as they are highly reactive.However, roots accumulate ROS at some level to control root growth [5].Hydrogen peroxide represses the expression of cell cycle-related genes in the meristematic zone [6].UPBEAT1 (UPB1), a bHLH domain-containing transcription factor, is a key regulator of the balance between cell proliferation and elongation by regulating the expression of several peroxidase genes.The spatial accumulation patterns of superoxide and hydrogen peroxide influence the size of the meristematic and elongation zones [7].MYB30 is an ROS-responsive transcription factor and MYB30 regulates root cell elongation under ROS and abscisic acid signaling [8,9].ANAC032 regulates MYB30 expression and a transcription network that modulates cell elongation [10].These results indicate that ROS regulates both cell proliferation and elongation in the root.Interestingly, MYB30 and ANAC032 are not direct targets of UPB1.
In the present study, we focused on the UPB1 gene regulatory network, which regulates primary root growth.Although UPB1 directly regulates the expression of several peroxidase genes, it also targets other genes.2,375 UPB1-responsive genes are identified by microarray analysis and 166 putative UPB1 direct targets are identified by ChIP-chip analysis [7].To elucidate UPB1 regulatory transcriptional network, we focused on the transcription factors among the 166 UPB1 direct targets.Among them, we chose a transcription factor, MYB50 whose function had not yet been analyzed.Functional analysis of MYB50 can provide insights into root growth regulation under the UPB1 gene regulatory network.We confirmed the transcriptional network from UPB1 to MYB50 by multi-color time-lapse imaging of A. thaliana roots.We also investigated MYB50 regulatory networks using RNA-seq and ChIP-seq analyses.Based on these analyses, we found that MYB50 directly regulated the expression of several cell wall modification genes.We further investigated the root phenotypes of the overexpressor of MYB50 target genes, especially PECTIN METHYLESTERASE INHIBITOR 8 (PMEI8).PMEI8 overexpression resulted in a shorter mature cell length in the root.These results indicate that UPB1 regulates root growth through MYB50 expression as a transcription factor for controlling further downstream gene expression and that MYB50 regulates cell elongation by controlling cell wall modification genes under the UPB1 transcription network.

Plant materials and growth conditions
Arabidopsis thaliana Columbia-0 (Col-0) was used as the wild-type.The T-DNA insertion line, upb1-1 was obtained from the SLAK collection and the seed stock center of the Arabidopsis Biological Resource Center (ABRC).upb1-1 mutant was genotyped using a left-border primer on T-DNA (LB), right-side primers on the genome (RP), and left-border primers on the genome (LP) as described previously [7].
All seeds were sterilized with 1% bleach and 0.05% Triton X-100 for 5 min and then washed thrice with sterilized water.Seeds were germinated on Murashige and Skoog (MS; FUJIFILM Wako Pure Chemical, Osaka, Japan) medium supplemented with 1% sucrose and 1% agar after two days at 4˚C.Plants were grown vertically in a chamber (Panasonic, Osaka, Japan) at 22˚C with 16-hour-light/8-hour-dark cycle.For estradiol treatment, 6-day-old seedlings were transferred onto MS agar plates containing 5 μM estradiol (FUJIFILM Wako Pure Chemical).

Plasmid construction and plant transformation
Genomic DNA from Columbia was used as a template for the amplification of the 3,020 bp upstream region of pMYB50 for promoter cloning.One base of 5' dA overhang was added to the PCR amplicon of pMYB50 using Taq polymerase (Takara Bio Inc., Shiga, Japan), which was then cloned into pENTR5'-TOPO (Thermo Fischer Scientific, Waltham, MA, USA) and named pENTR5'-pMYB50.For the genomic coding region and cDNA for MYB50 and PMEI8 cloning, MYB50 genomic and cDNA regions and the cDNA region of PMEI8 were amplified using the forward primer containing CACC sequences for TOPO cloning and the reverse primer.CACC-gMYB50, CACC-cMYB50, and CACC-cPMEI8 fragments were cloned into pENTR/D-TOPO (Thermo Fischer Scientific).For YFP-cMYB50, YFP-cPMEI8, YFP-cUPB1, and CFP-cUPB1 cloning, the cMYB50, cPMEI8, and cUPB1 cDNA regions were amplified using forward and reverse primers containing the BamHI site just before the MYB50, PMEI8, or UPB1 termination codon.The cMYB50-Bam HI, cPMEI8-Bam HI, and cUPB1-Bam HI fragments were cloned into Aor51H1, and the Bam HI sites of YFP-Aor51H1-Bam HI-pDONR201 plasmids [8] and CFP-Aor51H1-Bam HI-pDONR201 plasmids [11], respectively.

Quantitative real-time RT-PCR
RNA was isolated from the roots by using the RNeasy Plant Kit (QIAGEN, Hilden, Germany).For RNA isolation from meristematic and elongation zones, 6-day-old plants were dissected as previously described [7].First-strand cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO Co., Ltd., Osaka, Japan).Quantitative real-time RT-PCR (RT-qPCR) was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO) on a real-time PCR Eco system (PCRmax, Stone Staffordshire, UK).The sequences of primers used in this study are provided in S1 Table .The RT-qPCR efficiency and CT values were determined using standard curves for each primer set.The efficiency-corrected transcript values of three biological replicates for all samples were used to determine the relative expression values.Each value was normalized against the level of PDF2 [14].

RNA-seq experiments
Total RNA was isolated from roots; cDNA libraries were generated from 500 ng of total RNA using the NEBnext Ultra II RNA Library Prep kit (New England Biolabs, Ipswich, MA, USA), following the manufacturer's protocols.The ends of the cDNA libraries were sequenced for 60 cycles using a paired-end module on the Illumina NextSeq 500 platform (Illumina, San Diego, CA, USA).Two biological replicates were used in each experiment.

RNA-seq data analysis
Short-read sequencing results were mapped to the A. thaliana genome (TAIR10: www.arabidopsis.org/)using the Bowtie software [15].These datasets were normalized, and the false discovery rate (FDR) and fold change were calculated using the edgeR package for R [16].We used an FDR of q < 0.001 as the cut-off to determine differentially expressed genes.Data were deposited in the DNA Data Bank of Japan (DDBJ) sequence Read Archive (DRA) (https:// www.ddbj.nig.ac.jp/index-e.html)under accession number DRA016078.

ChIP-seq experiments
Over 1,200 plants per genotype were grown on MS medium for six days.These plants were then transferred onto MS medium containing 5 μM estradiol.One day after transfer, whole roots were fixed and flash-frozen using liquid nitrogen according to a previously described protocol [7].ChIP was performed as described previously [17] with anti-GFP antibody (ab290, Abcam, Cambridge, UK).ChIP DNA library was generated with a ChIP-Seq Sample Prep Kit (Illumina, San Diego, CA, USA) and the resulting DNA library was analyzed using GAII (Illumina) as described previously [17].

ChIP-seq data analysis
Base calling of sequence reads obtained by GAII was performed using the GAII pipeline software.Mapping of these sequence reads was performed using Bowtie [15] with default parameters.The resulting sequence alignment/map (SAM) file was converted into a binary alignment/ map (BAM) format file by Samtools 0.1.18[18].Significant ChIP DNA peaks (FDR q < 10 −20 ) were annotated as YFP-MYB50 binding loci using the Model-based Analysis of ChIP-Seq (MACS2) software [19], with the genome size parameter dm (1.2e 8 ).The forwardand reverse-peak distributions were validated using MACS2 and visualized using R (http:// www.R-project.org/).BAM and indexed BAM files were used to visualize mapping patterns using Integrative Genomics Viewer 2.13.0.Based on the sequence data of 1,000 bp upstream from the translation start site for each gene, obtained from the TAIR database (TAIR10), genes near each peak were identified using a local BLAST tool [20] with the following parameters: blastn -evalue 0.1 -outfmt 6. Manual curation was performed to remove incorrect annotations arising from similarities in promoter sequence regions.The ChIP-seq data were deposited in the DNA Data Bank of Japan (DDBJ) sequence Read Archive (DRA) (https://www.ddbj.nig.ac.jp/index-e.html)under accession number DRA016088.

Phenotyping and microscopy
To measure the whole root length, the roots were scanned using a flatbed scanner GT-7400U (Epson, Nagano, Japan) while growing on plates.The root length was measured by importing the scanned images into Fiji (imagej.net/software/fiji/). Prior to confocal imaging, roots were stained with propidium iodide (PI; FUJIFILM Wako pure chemical; 10 μg mL -1 in water) for 3 to 5 min.Confocal microscopy was performed using a Leica SP8 system (Leica Camera AG, Wetzlar, Germany) with 448 nm excitation and 460-510 nm emission for CFP, 488 nm excitation and 500-550 nm emission for GFP, 488 nm excitation and 490-543 nm emission for YFP, and 555 nm excitation and 580-680 nm emission for PI.Image assembly was performed using the LAS X software (Leica Camera AG).
For time-lapse imaging, Lab-Tek Chambered Coverglass w/cvr (Thermo Fisher Scientific) was used, as described previously [8].For time-lapse imaging of multi-color reporter line, 6-day-old seedlings of pXVE::CFP-cUPB1/pMYB50::gMYB50-GFP/upb1-1 in MS medium were placed on chambered cover glass in media containing 5 μM estradiol.The chambers containing plants were imaged using a Leica SP8 confocal microscope at objectives of 20×.Timelapse images were captured using LAS X every 40 min for 7 h and 20 min.Images were also assembled with LAS X.To quantify CFP-cUPB1 and gMYB50-GFP intensities, 50 z-stack images of the roots were taken with LAS X every 20 min for 5 h using 40× (oil) objectives.Of the 50 z-stack images for each time point, the image with the sharpest focus on the nucleus of the cortex or epidermal cells was selected, and CFP and GFP intensities were quantified as a mean intensity using Fiji software.
To measure the increase in root length, 4-day-old seedlings in MS medium were placed on chambered cover glass in media containing 5 μM estradiol.The chambers containing the plants were imaged using a DMI 6000 B-AFC fluorescence microscope (Leica Camera AG) under a 20× objective lens.Time-lapse images were captured every 30 min for 24 h.The increase in root length was measured using the Fiji software on a time series consisting of images taken every 30 min.

Statistical analysis
All statistical analyses were performed using Microsoft Excel or R. Hypergeometric testing for overlapping genes between ChIP and RNA-seq was performed using the online program http://nemates.org/MA/progs/overlap_stats.html.Details of the analyses are provided in the figure legends.

UPB1 regulates MYB50 expression at the elongation zone
To confirm previous results [7], we analyzed MYB50 expression in the upb1-1 mutant by RT-qPCR by using RNA extracted separately from meristematic and elongation zones.Proper dissection was confirmed by assessing the expression of zone-specific genes CYCB1;1 and UPB1, which are predominantly expressed in the meristematic and elongation zones, respectively (Fig 1A).MYB50 expression was significantly higher in the elongation zone of the upb1-1 mutant than in Col-0 plants (Fig 1A).Consistent with previous results [7], UPB1 weakly suppressed MYB50 expression in the elongation zone.We constructed an estradiol-inducible YFP-UPB1 transformant (pXVE::YFP -UPB1).Following 24 h of 5 μM estradiol treatment, UPB1 expression substantially increased, resulting in a statistically significant 50% reduction in MYB50 expression (Fig 1B).Time-lapse imaging was performed every 30 min after estradiol treatment for 24 h to measure pXVE::YFP-UPB1 root elongation.After estradiol treatment, elongation of pXVE::YFP-UPB1 roots was inhibited by about a factor of two compared to Columbia (Fig 1C).In addition to time-lapse imaging, we measured the root length of UPB1 overexpressor on vertical plates containing 5 μM estradiol at 24, 48, and 72 h (Fig 1D).After 72 h of estradiol treatment, the increase in root length in UPB1 overexpressor was reduced to approximately 70% of that in the Columbia.These findings indicate that UPB1 overexpression repressed MYB50 and led to a reduction in root growth, similar to previous results [7].
We examined MYB50 expression patterns in roots by creating a translational fusion reporter (pMYB50::gMYB50-GFP) expressed in both in Columbia and upb1-1.In Columbia, we observed MYB50-GFP fluorescence in the early differentiation zone but not in the meristematic and the beginning of the apparent elongation zone (Fig 2A

MYB50 regulates the length of the meristematic zone and mature cell length
After showing that MYB50 expression is repressed by UPB1, we next investigated how MYB50 is involved in root growth.We attempted to analyze the MYB50 function in root growth using genetic methods but an MYB50 T-DNA insertion line is not available from the ABRC seed stock center.To investigate the effects of MYB50 overexpression on root growth, we made a line expressing YFP-MYB50 under the control of an estradiol-inducible promoter (pXVE:: YFP-MYB50).In this inducible line, MYB50 expression reached more than 100 times the Columbia level in both independent lines after 24 h of estradiol treatment (Fig 3A).We investigated the root growth phenotype caused by induced MYB50 with time-lapse imaging every 30 min for 24 h.After 24 h of induction with 5 μM estradiol, root elongation of pXVE::YFP-MYB50 was inhibited (Fig 3B).In addition to time-lapse imaging, we measured the root length of MYB50 overexpressors on the vertical plates containing 5 μM estradiol at 24, 48, and 72 h (Fig 3C).After 72 h of estradiol treatment, the root length increments of MYB50 overexpressors decreased to approximately 40% of that of the Columbia.
To determine the detailed effects of MYB50 on root growth, we counted the number of cortical cells in the meristematic zone and measured the mature cell length in MYB50 induced-and upb1-1, p = 0.0096.(B) UPB1 and MYB50 expression analysis of roots of 6-day old seedlings in pXVE::YFP-UPB1/Columbia with and without 5 μM estradiol (Est) treatment for 24 h measured by RT-qPCR (n = 5).Statistically significant differences were determined using Student's t-test compared with and without estradiol treatment (*** p < 0.001; * p < 0.05).p value: comparison of UPB1 expression, p < 0.001; comparison of MYB50 expression, p = 0.016.(C) Root length in 4-day-old seedlings treated with 5 μM estradiol within the agar medium.Root length was measured every 30 min using time-lapse imaging (n = 5, means ± SD). (D) Root length in 4-day-old seedlings grown vertically on agar medium containing 5 μM estradiol.Root length was measured every 24 h for 72 h (Columbia, n = 38; pXVE::YFP-UPB1, n = 25, means ± SD).To assess statistical significance, we employed Student's t-test for comparisons between Columbia and transgenic at each time (*** p < 0.001).
https://doi.org/10.1371/journal.pone.0285241.g001overexpressor.Two independent lines of pXVE::YFP-MYB50 treated with 5 μM estradiol for 24 h showed reduced cortical cell numbers in the meristematic zone and shorter mature cell length (Fig 3D -3G).Taken together, these results suggest that MYB50 overexpression negatively regulates meristematic zone size and mature cell length, similar to UPB1 overexpression.Because UPB1 is known to control ROS homeostasis at the root tip, we investigated whether MYB50 expression is induced by ROS.A 24 h treatment with H 2 O 2 did not affect MYB50 expression in wild type roots (S4A Fig) .In addition to this, we investigated how the inhibition of root elongation under MYB50 over-expression interacted with ROS-controlled root growth.H 2 O 2 , a ROS, is known to inhibit root elongation [22].A combined treatment with estradiol and 500 μM H 2 O 2 resulted in a smaller meristem size than estradiol treatment alone in Columbia roots (S4B and S4C Fig) .Interestingly, MYB50 induced-overexpressor showed smaller meristem sizes than Columbia plants in the estradiol treatment but even smaller meristem sizes upon H 2 O 2 and estradiol treatment (S4B and S4C Fig) .This additive effect suggests that the MYB50 could inhibit root growth independent of ROS regulation.
To identify MYB50 direct targets, we performed ChIP-seq analysis using 24 h estradioltreated pXVE::YFP-MYB50.Sequence reads were mapped to the A. thaliana genome TAIR10 (https://www.arabidopsis.org/index.jsp) and genomic regions bound by MYB50 according to an FDR q-value of < 10 −20 in the dataset (S1 Dataset).ChIP-seq analysis identified 2,639 regions 1,000 bp upstream of the translation initiation site of genes.Of course, this number included false-positive genes because of the technical limitations of the ChIP-seq protocol.To find MYB50-biological direct target genes, we combined RNA-seq and ChIP-seq data.We searched for common genes between MYB50-bound genes in ChIP-seq and total genes that were upregulated (368 genes) or downregulated (151 genes) at least one time point during estradiol induction of MYB50 in time-course RNA-seq.A statistically significant relationship was observed between 50 common genes and MYB50 bound genes (hypergeometric probability test, p = 3.23e -07 ), conversely, no such relationship was observed between six common channel, Right panels: GFP channels.Scale bars, 5 μm.(C) Quantification of CFP-UPB1 and MYB50-GFP intensity after estradiol treatment (solid lines) and no-estradiol treatment (dashed lines) every 20 min.Each signal intensity was measured every 20 min using time-lapse imaging (n = 4: two nuclei from each of the two roots, means ± SD). https://doi.org/10.1371/journal.pone.0285241.g002and 4C).This suggests that MYB50 preferentially upregulates genes bound to MYB50 in the promoter region.

MYB50 regulates the expression of pectin modification genes
Among the MYB50 direct target genes previously identified as differentially expressed in upb1-1 [7], we focused on PMEI8 (At3g17130) and its role in root growth.PMEIs are known to regulate pectin status, and PMEI8 overexpression partially complements the short-root phenotype in cobra mutants [23].However, the detailed effects of PMEI8 overexpression on root growth have not been reported.In silico analysis of RootMap gene expression data [24] showed that PMEI8 is highly expressed in sections 7 to 9 (from the elongation zone close to the early differentiation zone) (S7 Fig) .Moreover, the expression of MYB50 in the RootMap dataset peaks at section 7, consistent with the idea that MYB50 regulates PMEI8 expression in the elongation and early differentiation zones (Fig 2A).In addition, MYB50 bound approximately 1,000 bp upstream of the PMEI8 coding region according to our ChIP-seq data (Fig 5A).As PMEI8 was upregulated in the MYB50 induced-overexpressor (Fig 5B ), we decided to investigate the phenotype of an overexpressor of PMEI8.
To investigate how PMEI8 overexpression affected root growth, we made a YFP-PMEI8 inducible line (pXVE::YFP-PMEI8) that expressed PMEI8 more than 4,000 times after 24 h of estradiol treatment (Fig 5C).Time-lapse imaging was performed every 30 min after 5 μM estradiol treatment to measure root elongation.After 24 h estradiol treatment, elongation of pXVE::YFP-PMEI8 was approximately 70% compared to Columbia (Fig 5D).We also measured the root length of PMEI8 overexpressors on the vertical plates containing 5 μM estradiol every 24 h for a duration of 72 h.After 48 and 72 h of induction with estradiol, root lengths of PMEI8 overexpressors were slightly shortened compared to Columbia (Fig 5E).We measured the number of cortical cells in the meristematic zone and the mature cell length in the PMEI8 https://doi.org/10.1371/journal.pone.0285241.g003induced-overexpressors.Both the cell numbers in the meristematic zone of no-estradiol and estradiol-treated pXVE::YFP-PMEI8 plants were slightly reduced compared to those of Columbia (#1 was statistically significant but #2 was not statistically significant).Estradiol-treated pXVE::YFP-PMEI8 plants showed significantly shorter mature cell length than control cells  -5I).The mature cell length phenotype was consistent with MYB50 induced-overexpressor in which MYB50 directly upregulated PMEI8 expression (Fig 5A and 5B).This result indicates that PMEI8 is a negative regulator of root growth, especially cell length, under the MYB50 transcriptional regulation.

MYB50 is a direct downstream target of UPB1
In a previous study, UPB1 was shown to regulate root development through reactive oxygen species (ROS) homeostasis at the root tip by directly controlling the expression of peroxidase genes [7].In the study, UPB1 is shown to bind to the promoter regions of several transcription factor genes that are probably not involved in ROS homeostasis.We hypothesized that UPB1-targeted transcription factors also regulate root growth through a regulatory network independent of ROS.Among these, we were interested in a transcription factor whose function has not yet been reported.In this study, we selected MYB50 as a candidate gene that is part of the UPB1 regulatory network for root growth.MYB50 expression was upregulated in the upb1-1 mutant but downregulated in the UPB1 overexpressor.As MYB50 is expressed in the elongation zone, MYB50 is likely to have a role in the root development.Similarly, an MYB50 induced-overexpressor showed decreased root elongation compared to the control.These results indicate that MYB50 is a root growth inhibitor.We confirmed UPB1-MYB50 transcriptional regulation by multi-color reporter lines.Visualizing transcriptional regulation is challenging; however, recent imaging technologies have allowed us to achieve this goal.MYB50-GFP expression was repressed after the estradiol induction of CFP-UPB1 in root cells.This system might be used not only for other gene regulatory networks of transcription factors but also for tracing alterations in the subcellular localization of several proteins simultaneously.

MYB50 regulated PMEI8 expression under the UPB1 gene regulatory network
Transcriptome analysis of MYB50 induction indicated that MYB50 did not regulate the expression of ROS homeostasis genes regulated by UPB1.In support of this, H 2 O 2 treatment had an additive effect on the reduction in meristem size in the MYB50 overexpressor.
Columbia and pXVE::YFP-MYB50 #2, p = 0.00702.(C) PMEI8 expression in 6-day-old roots after 24 h of 5 μM estradiol treatment as measured using RT-qPCR (n = 3, mean ± SD).Statistically significant difference was determined using Student's ttest compared to Columbia (*** p < 0.001).(D) Root length in 4-day-old seedlings treated with 5 μM estradiol within the agar medium.Root length was measured every 30 min for 24 h using time-lapse imaging (n = 5, means ± SD). (E) Root length in 4-day-old seedlings grown vertically on agar medium containing 5 μM estradiol.Root length was measured every 24 h for 72 h (Columbia, n = 53; pXVE::YFP-PMEI8 #1, n = 33; pXVE::YFP-PMEI8 #2, n = 32; means ± SD).Statistically significant differences were determined using Student's t-test by comparing Columbia and pXVE::YFP-PMEI8 at each time (*** p < 0.001).Interestingly, the number of common MYB50 and UPB1-regulated genes was low.As the MYB50 expression level by the pXVE promoter was much stronger than that in upb1-1 mutants, MYB50 overexpression affected its downstream genes more than the UPB1 gene regulatory network.However, we found one interesting gene, PMEI8, among MYB50 target genes which was also derepressed in the upb1-1 mutant.Our results showed that PMEI8 under the MYB50 gene regulatory network inhibited cell elongation and consequently shortened mature cell length.
Fine-tuning of pectin methyl esterification is considered important for cell wall stiffening, which regulates cell growth [25,26].Spatiotemporal control of pectin methyl esterification is affected by PMEs and PMEIs in A. thaliana.Although PMEI family proteins are predicted to have the same function, each PMEI, when knocked out or over-expressed, can give rise to different root phenotypes.PMEI-1, PMEI-2, or PMEI9 overexpression resulted in a longer primary root phenotype, whereas PMEI4 overexpression resulted in a short root phenotype [27,28].These results on PMEI function support the hypothesis that spatiotemporally controlled PMEIs regulate root growth.Moreover, overexpression of another PMEI13 (At5g62360) did not alter root length; however, overexpression of that PMEI13 with EXPA5, which is considered a cell wall-loosening enzyme, increased root length [29].
PMEI8 has been identified as a partial suppressor of cobra mutations.PMEI8 overexpression in cobra mutants can ameliorate their root growth defects [23].According to these functions, there are coregulatory genes of PMEI8 in the MYB50 and UPB1 regulatory gene networks.
The data presented here, combined with previous UPB1 data, are shown as a model of the gene regulatory network (Fig 6).UPB1 negatively impacts meristem size through ROS homeostasis by repressing several peroxidase genes, which are associated with larger meristems when overexpressed [7].In our study, we demonstrated that UPB1 directly represses MYB50 in the elongation zone.MYB50, in turn, negatively regulates meristem size, with the MYB50-induced overexpressor displaying smaller meristem size and mature cell length (Fig 3E and 3F).
The mechanism behind MYB50's suppression of meristem size remains unclear.Concerning cell elongation, we found that MYB50 suppresses it through the transcriptional activation of downstream genes, one of which is PMEI8.In summary, the integration of both UPB1 and MYB50 transcriptional networks indicates that UPB1 not only controls ROS homeostasis but also influences cell wall status, particularly pectin modification via PMEI8, a direct target of MYB50.However, the regulation of PMEI8 alone does not entirely elucidate the complex regulatory mechanisms of root growth within the MYB50 network.To support this hypothesis, we identified several cell wall modification genes in the transcriptome datasets.To fully understand the MYB50 gene regulatory network that controls root growth, we need to investigate the gene functions in the near future.
We found that 368 genes were significantly upregulated in the RNA-seq analysis of the MYB50 induction line.Among the upregulated genes, we identified GO terms representing the responses to abiotic stress and chemical compounds (Fig 4A).Furthermore, we combined these data with ChIP-seq data from YFP-MYB50, concluded that MYB50 was a transcriptional activator, and identified 50 genes as MYB50 direct target genes.We identified ABA repressor 1 (ABR1), NAC004, ERF019, and CBF2 [30,31].These transcription factors are all involved in the abscisic acid (ABA) response, especially in stress responses such as cold and drought.In addition to these transcription factors, we identified ABA and stress response marker genes such as LEA, RD29A, ERD4, ERD10, and COR47 [32][33][34][35][36][37][38].These results indicated that MYB50 may be involved in plant stress responses regulated by ABA.In future studies, to deepen our understanding of the function of MYB50 in ABA signaling, we need to investigate the phenotypes of MYB50 overexpressor under abiotic stress.

Conclusions
In this study, we identified a transcription factor, MYB50, that is one of downstream genes of UPB1 for regulating root growth.We demonstrated MYB50's direct role in inducing the expression of PMEI8, which participates in cell wall modification and influences root growth.While UPB1 regulates root growth through maintaining reactive oxygen species (ROS) homeostasis at the root tip, our findings suggest that MYB50, by controlling PMEI8 expression, also contributes to root growth regulation by influencing cell wall status.Root growth is (EZ): Elongation zone.UPB1 directly represses PER57 in the TZ and negatively regulates the meristem size [7].In the EZ, UPB1 directly represses MYB50 expression.The root phenotypic analysis using pXVE::YFP-MYB50 revealed that MYB50 negatively regulates the meristem size through a pathway separate from ROS regulation, whereas the cause remains unknown.However, MYB50 was expressed in the EZ close to the differentiation zone and directly activated PMEI8 expression.MYB50 regulatory network and PMEI8 inhibit cell elongation as pXVE:: YFP-MYB50 and pXVE::YFP-PMEI8 shortened mature cell length.The MYB50-regulated transcriptional network includes several cell wallassociated genes, which may coregulate with PMEI8 to exert some coregulation.https://doi.org/10.1371/journal.pone.0285241.g006complex, and the function of a single transcription factor alone is insufficient to provide a comprehensive explanation.The functional analysis of MYB50 underscores the intricate interplay between the ROS regulatory pathway and the cell wall modification system via PMEI8, regulated by MYB50 downstream of UPB1, which is vital for normal root development.However, PMEI8 regulation alone does not fully elucidate the regulatory mechanisms of root growth in the MYB50 network.As we also found several transcription factors in the MYB50 direct target genes, further analysis of their functions will provide more comprehensive insights into the regulatory mechanisms of root growth within the MYB50 gene regulatory network.
and S1 Fig).However, in the upb1-1 mutant, weak MYB50-GFP fluorescence emerged from the middle of the apparent elongation zone.In both backgrounds, towards the mature region of the root, we noticed fluorescence appeared in large puncta, probably corresponding to nuclei (Fig 2A and S1 Fig), consistent with UPB1 regulating MYB50 in the elongation zone.Next, we performed time-lapse

Fig 1 .
Fig 1. UPB1 regulates MYB50 expression at the elongation zone.(A) UPB1, MYB50, and CYCB1;1 expression in both of the meristematic and elongation zones in roots of 7-day old seedlings of Columbia (Col-0; gray) and upb1-1 (pink) as measured by RT-qPCR (n = 5).MZ and EZ indicate the meristematic and elongation zone, respectively.Bars show mean ± SD.Statistically significant differences were determined using Student's t-test by comparing Columbia and upb1-1 in each zone (*** p < 0.001; ** p < 0.01).p value: comparison of UPB1 expression in MZ of Columbia and upb1-1, p = 0.002; comparison of UPB1 expression in EZ of Columbia and upb1-1, p < 0.001; comparison of MYB50 expression in EZ of Columbia

Fig 2 .
Fig 2. MYB50 expression pattern in the primary root and the multi-color fluorescent protein expression of CFP-UPB1 and MYB50-GFP.(A) Confocal microscopic images of primary root tip of 7-day old seedlings.White arrow heads indicate the nuclei with apparent GFP fluorescence.Scale bars, 100 μm.(B) Timepoint images every 60 min in the visualization of transcriptional repression of MYB50 by UPB1 in one nucleus.6-dayold seedlings were stained by propidium iodide (PI) and CFP-UPB1 and each MYB50-GFP signal was independently detected.Apparent nuclei exhibiting detectable CFP or GFP fluorescence were visualized and enlarged.Left panels: merged images of CFP, GFP, and PI, Middle panels: CFP

Fig 6 .
Fig 6.A schematic model of gene regulatory network under UPB1 and MYB50 control.Black arrows and "T"s indicate transcriptionally direct target genes.The outline arrow and "T"s indicate an effect on the root phenotype.(MZ): Meristematic zone.(TZ): Transition zone.(EZ):Elongation zone.UPB1 directly represses PER57 in the TZ and negatively regulates the meristem size[7].In the EZ, UPB1 directly represses MYB50 expression.The root phenotypic analysis using pXVE::YFP-MYB50 revealed that MYB50 negatively regulates the meristem size through a pathway separate from ROS regulation, whereas the cause remains unknown.However, MYB50 was expressed in the EZ close to the differentiation zone and directly activated PMEI8 expression.MYB50 regulatory network and PMEI8 inhibit cell elongation as pXVE:: YFP-MYB50 and pXVE::YFP-PMEI8 shortened mature cell length.The MYB50-regulated transcriptional network includes several cell wallassociated genes, which may coregulate with PMEI8 to exert some coregulation.